Fingering Instabilities in Dewetting Nanofluids

The growth of fingering patterns in dewetting nanofluids (colloidal solutions of thiol-passivated gold nanoparticles) has been followed in real time using contrast-enhanced video microscopy. The fingering instability on which we focus here arises from evaporatively-driven nucleation and growth a nanoscopically thin "precursor" solvent film behind the macroscopic contact line. We find that well-developed isotropic fingering structures only form for a narrow range of experimental parameters. Numerical simulations, based on a modification of the Monte Carlo approach introduced by Rabani et al. [Nature 426, 271 (2003)], reproduce the patterns we observe experimentally

A Study on the Dependence of Water Tree Permittivity with Time

During the growth of water trees in the insulation of a cable the distribution of the electric field is modified because of the local change of the dielectric properties of the material. It results a local enhancement of the electric field which could increase the risk of breakdown. The key factor is the permittivity of the water tree and the aim of the work is to determine its possible values and, particularly, the law of its increase with time during the of the trees. The paper presents permittivity measurements in uniform field in MV and powe

Towards quantitative molecular mapping of cells by Raman microscopy: Using AFM for decoupling molecular concentration and cell topography

Raman micro-spectroscopy (RMS) is a non-invasive technique for imaging live cells in vitro. However, obtaining quantitative molecular information from Raman spectra is difficult because the intensity of a Raman band is proportional to the number of molecules in the sampled volume, which depends on the local molecular concentration and the thickness of the cell. In order to understand these effects, we combined RMS with atomic force microscopy (AFM), a technique that can measure accurately the thickness profile of the cells. Solution-based calibration models for RNA and albumin were developed to create quantitative maps of RNA and proteins in individual fixed cells. The maps were built by applying the solution-based calibration models, based on partial least squares fitting (PLS), on raster-scan Raman maps, after accounting for the local cell height obtained from the AFM. We found that concentrations of RNA in the cytoplasm of mouse neuroprogenitor stem cells (NSCs) were as high as 25 ± 6 mg ml-1, while proteins were distributed more uniformly and reached concentrations as high as ∼50 ± 12 mg ml-1. The combined AFM-Raman datasets from fixed cells were also used to investigate potential improvements for normalization of Raman spectral maps. For all Raman maps of fixed cells (n = 10), we found a linear relationship between the scores corresponding to the first component (PC1) and the cell height profile obtained by AFM. We used PC1 scores to reconstruct the relative height profiles of independent cells (n = 10), and obtained correlation coefficients with AFM maps higher than 0.99. Using this normalization method, qualitative maps of RNA and protein were used to obtain concentrations for live NSCs. While this study demonstrates the potential of using AFM and RMS for measuring concentration maps for individual NSCs in vitro, further studies are required to establish the robustness of the normalization method based on principal component analysis when comparing Raman spectra of cells with large morphological differences

Cytoplasmic RNA in undifferentiated neural stem cells: A potential label-free raman spectral marker for assessing the undifferentiated status

Raman microspectroscopy (rms) was used to identify, image, and quantify potential molecular markers for label-free monitoring the differentiation status of live neural stem cells (NSCs) in vitro. Label-free noninvasive techniques for characterization of NCSs in vitro are needed as they can be developed for real-time monitoring of live cells. Principal component analysis (PCA) and linear discriminant analysis (LDA) models based on Raman spectra of undifferentiated NSCs and NSC-derived glial cells enabled discrimination of NSCs with 89.4% sensitivity and 96.4% specificity. The differences between Raman spectra of NSCs and glial cells indicated that the discrimination of the NSCs was based on higher concentration of nucleic acids in NSCs. Spectral images corresponding to Raman bands assigned to nucleic acids for individual NSCs and glial cells were compared with fluorescence staining of cell nuclei and cytoplasm to show that the origin of the spectral differences were related to cytoplasmic RNA. On the basis of calibration models, the concentration of the RNA was quantified and mapped in individual cells at a resolution of ∼700 nm. The spectral maps revealed cytoplasmic regions with concentrations of RNA as high as 4 mg/mL for NSCs while the RNA concentration in the cytoplasm of the glial cells was below the detection limit of our instrument (∼1 mg/mL). In the light of recent reports describing the importance of the RNAs in stem cell populations, we propose that the observed high concentration of cytoplasmic RNAs in NSCs compared to glial cells is related to the repressed translation of mRNAs, higher concentrations of large noncoding RNAs in the cytoplasm as well as their lower cytoplasm volume. While this study demonstrates the potential of using rms for label-free assessment of live NSCs in vitro, further studies are required to establish the exact origin of the increased contribution of the cytoplasmic RNA. © 2012 American Chemical Society

Raman spectroscopy study of curvature-mediated lipid packing and sorting in single lipid vesicles

Cellular plasma membrane deformability and stability is important in a range of biological processes. Changes in local curvature of the membrane affects the lateral movement of lipids, affecting the biophysical properties of the membrane. An integrated holographic optical tweezers (HOT) and Raman microscope was used to investigate the effect of curvature gradients induced by optically stretching individual giant unilamelar vesicles (GUV) on lipid packing and lateral segregation of cholesterol in the bilayer. The spatially-resolved Raman analysis enabled detection of induced phase separation and changes in lipid ordering in individual GUVs. Using deuterated cholesterol, the changes in lipid ordering and phase separation were linked to lateral sorting of cholesterol in the stretched GUVs. Stretching the GUVs in the range of elongation factors 1-1.3 led to an overall decrease in cholesterol concentration at the edges compared to the centre of stretched GUVs. The Raman spectroscopy results were consistent with a model of the bilayer accounting for cholesterol sorting in both bilayer leaflets, with a compositional asymmetry of 0.63±0.04 in favour of the outer leaflet. The results demonstrate the potential of the integrated HOT-Raman technique to induce deformations to individual lipid vesicles and to simultaneously provide quantitative and spatially-resolved molecular information. Future studies can extend to include more realistic models of cell membranes and potentially live cells

Surface-enhanced Raman spectroscopy of the endothelial cell membrane

We applied surface-enhanced Raman spectroscopy (SERS) to cationic gold-labeled endothelial cells to derive SERS-enhanced spectra of the bimolecular makeup of the plasma membrane. A two-step protocol with cationic charged gold nanoparticles followed by silver-intensification to generate silver nanoparticles on the cell surface was employed. This protocol of post-labelling silver-intensification facilitates the collection of SERS-enhanced spectra from the cell membrane without contribution from conjugated antibodies or other molecules. This approach generated a 100-fold SERS-enhancement of the spectral signal. The SERS spectra exhibited many vibrational peaks that can be assigned to components of the cell membrane. We were able to carry out spectral mapping using some of the enhanced wavenumbers. Significantly, the spectral maps suggest the distribution of some membrane components are was not evenly distributed over the cells plasma membrane. These results provide some possible evidence for the existence of lipid rafts in the plasma membrane and show that SERS has great potential for the study and characterization of cell surfaces

Rapid production of human liver scaffolds for functional tissue engineering by high shear stress oscillation-decellularization

The development of human liver scaffolds retaining their 3-dimensional structure and extra-cellular matrix (ECM) composition is essential for the advancement of liver tissue engineering. We report the design and validation of a new methodology for the rapid and accurate production of human acellular liver tissue cubes (ALTCs) using normal liver tissue unsuitable for transplantation. The application of high shear stress is a key methodological determinant accelerating the process of tissue decellularization while maintaining ECM protein composition, 3D-architecture and physico-chemical properties of the native tissue. ALTCs were engineered with human parenchymal and non-parenchymal liver cell lines (HepG2 and LX2 cells, respectively), human umbilical vein endothelial cells (HUVEC), as well as primary human hepatocytes and hepatic stellate cells. Both parenchymal and non-parenchymal liver cells grown in ALTCs exhibited markedly different gene expression when compared to standard 2D cell cultures. Remarkably, HUVEC cells naturally migrated in the ECM scaffold and spontaneously repopulated the lining of decellularized vessels. The metabolic function and protein synthesis of engineered liver scaffolds with human primary hepatocytes reseeded under dynamic conditions were maintained. These results provide a solid basis for the establishment of effective protocols aimed at recreating human liver tissue in vitro

Surface-enhanced Raman spectroscopy study of 4-ATP on gold nanoparticles for basal cell carcinoma fingerprint detection

The surface-enhanced Raman signals of 4-aminothiophenol (4-ATP) attached to the surface of colloidal gold nanoparticles with size distribution of 2 to 5 nm were used as a labeling agent to detect basal cell carcinoma (BCC) of the skin. The enhanced Raman band at 1075 cm-1 corresponding to the C-S stretching vibration in 4-ATP was observed during attachment to the surface of the gold nanoparticles. The frequency and intensity of this band did not change when the colloids were conjugated with BerEP4 antibody, which specifically binds to BCC. We show the feasibility of imaging BCC by surface-enhanced Raman spectroscopy, scanning the 1075 cm-1 band to detect the distribution of 4ATP-coated gold nanoparticles attached to skin tissue ex vivo

A study of Docetaxel-induced effects in MCF-7 cells by means of Raman microspectroscopy

Chemotherapies feature a low success rate of about 25%, and therefore, the choice of the most effective cytostatic drug for the individual patient and monitoring the efficiency of an ongoing chemotherapy are important steps towards personalized therapy. Thereby, an objective method able to differentiate between treated and untreated cancer cells would be essential. In this study, we provide molecular insights into Docetaxel-induced effects in MCF-7 cells, as a model system for adenocarcinoma, by means of Raman microspectroscopy combined with powerful chemometric methods. The analysis of the Raman data is divided into two steps. In the first part, the morphology of cell organelles, e.g. the cell nucleus has been visualized by analysing the Raman spectra with k-means cluster analysis and artificial neural networks and compared to the histopathologic gold standard method hematoxylin and eosin staining. This comparison showed that Raman microscopy is capable of displaying the cell morphology; however, this is in contrast to hematoxylin and eosin staining label free and can therefore be applied potentially in vivo. Because Docetaxel is a drug acting within the cell nucleus, Raman spectra originating from the cell nucleus region were further investigated in a next step. Thereby we were able to differentiate treated from untreated MCF-7 cells and to quantify the cell–drug response by utilizing linear discriminant analysis models

Quantitative single cell monitoring of protein synthesis at subcellular resolution using fluorescently labeled tRNA

We have developed a novel technique of using fluorescent tRNA for translation monitoring (FtTM). FtTM enables the identification and monitoring of active protein synthesis sites within live cells at submicron resolution through quantitative microscopy of transfected bulk uncharged tRNA, fluorescently labeled in the D-loop (fl-tRNA). The localization of fl-tRNA to active translation sites was confirmed through its co-localization with cellular factors and its dynamic alterations upon inhibition of protein synthesis. Moreover, fluorescence resonance energy transfer (FRET) signals, generated when fl-tRNAs, separately labeled as a FRET pair occupy adjacent sites on the ribosome, quantitatively reflect levels of protein synthesis in defined cellular regions. In addition, FRET signals enable detection of intra-populational variability in protein synthesis activity. We demonstrate that FtTM allows quantitative comparison of protein synthesis between different cell types, monitoring effects of antibiotics and stress agents, and characterization of changes in spatial compartmentalization of protein synthesis upon viral infection